Posted
by
Zonk
on Friday June 29, 2007 @07:43AM
from the a-little-hunter-killer-nanotech-goes-a-long-way dept.

Chris writes "German scientists have succeeded in snipping HIV out of human cellsafter it has integrated itself into a patient's DNA. The procedure is a breakthrough in bio-technology and fuels hope of a cure for AIDS. The group is only cautiously optimistic, though, as treating a full-on infection would be substantially different than succeeding in a controlled lab environment. 'Researchers ... began with the bacterial enzyme Cre recombinase, which exchanges any two pieces of DNA flanked on either end by a certain pattern of nucleotides (DNA subunits) known as loxP. HIV does not naturally contain loxP sites, so the team created a hybrid of the two DNA molecules, which they used to select a series of mutated Cre enzymes that were increasingly able to recognize the combined DNA. The final enzyme, Tre, removed all traces of HIV from cultured human cervical cells after about three months, the researchers report online today in Science.'"

Alright, I must be crazy. I was just thinking about HIV in the shower, and a similar idea came to my mind. Now it wasn't identical-that would have been freaky-but similar enough to make my hair stand up when I read the first/. entry this morning

Do you, by any chance, wake up to public radio or similar? I find that sometimes the first 10 minutes or so of what is said on the radio, before I become fully cognizant, gets absorbed into my subconscious so that I think it is weird when I hear the same bit of news later in the day.

HIV does not naturally contain loxP sites, so the team created a hybrid of the two DNA molecules, which they used to select a series of mutated Cre enzymes that were increasingly able to recognize the combined DNA. The final enzyme, Tre, removed all traces of HIV from cultured human cervical cells after about three months, the researchers report online today in Science.

When "a series of mutated" forms of X are "select[ed for,]" resulting in a "final" Y, that's evolution, not contrivance. The initial cutting with Cre was contrived; Tre is a new enzyme which doesn't need the LoxP sites and recognizes HIV as it is.

Basically, they played that "You have 5 steps to change NET to PAWN changing/adding/removing one letter each time: NET NEW SEW SAW PAW PAWN" game with an enzyme.

I read about this in PhysOrg [physorg.com] yesterday and they speak more about something the last paragraph of Scientific American only mentions. The fact that they wouldn't use this enzyme to remove HIV infections but instead to figure out which cells have been infected. The biggest problem in treating HIV is that it can go dormant and undetected for so long during which the host can infect others. It sounds horrible, but even being able to destroy all the cells infected with the virus is worth something though it may often prove fatal to the host. I don't think this is a 'cure' or 'vaccine' merely something that makes HIV treatments much much more effective.

Yes, and I'm happy to see that at least we're making *some* progress. I'm also saddened to see that apart from HIV, there is hardly any research going on to find cures for infectious diseases (TB, Malaria, viral diseases), at least when compared to the obscene amount of money being thrown into chronic or "lifestyle" diseases like diabetes and hypertension. This is all the more disturbing considering that infectious diseases afflict and kill so many more people than chronic diseases. It just so happens that most of the people afflicted happen to be from developing or poor countries, and hence, are not the target market segment for big pharma.

An interesting idea that I read somewhere proposed the setting up of Ansari-X style rewards or competitions for the company or team that first finds a cure/vaccine for these unfashionable diseases. This also becomes an easy way out for charity foundations like the Gates foundation, who're actually trying to do something meaningful in this field. Instead of giving grants to researchers much like a venture capitalist, perhaps instituting sizable multi-million dollar rewards is a better incentive for researchers. Plus, there is no need to monitor the charity money to make sure that it is being utilized properly. But then again, this might simply be an oversimplified solution to the problem.

Well, given our "lifestyle" isn't really changing (but actually getting worse), I'd say that soon more people will die due to their "living circumstances" (i.e. unhealthy food and lack of any kind of movement that doesn't require kicking the throttle pedal) than to diseases. If that didn't happen long ago.This aside, I would suggest your idea of "money for results" movement, but realize that research ain't something you can do in a garage with a few bucks of your spare money. You first of all have to throw

The research for new medications usually result in expensive drugs so they are usually targeted at a first-world market. A lot of R&D is also done to lower the cost of known medication that could prevent millions of deaths but that are just too expensive today.

Sadly, it's because the viral diseases you mentioned tend to happen to poor people, where as the middle and upper classes get the lifestyle diseases. Sad fact of the world is that people tend to do what gathers them the largest amount of resources most easily and fastest. This includes but it not limited to global warming and wars as well. We're talking billions here, not the hundreds of thousands, or even millions that philanthropic organizations could award for prizes. Limited manpower and limited resour

Sadly, it's because the viral diseases you mentioned tend to happen to poor people, where as the middle and upper classes get the lifestyle diseases.

Garbage. Viruses don't care about class barriers and eventually become problems for people of all income levels. Further, a large number of even the early AIDS victims were quite rich. As for federal funding, the "great unwashed" are a LARGE pool of voters.

The major impediment to vaccine research and production is legal and political attacks. It's too much

If medical care were socialized, there'd be less of a lure to develop so many "useless" medicines, and more of a lure to develop live saving medicine

Sorry, you're wrong. (parts of) Europe and canada have socialized medicine, and they're still not researching the TB vaccine. It's not an incentive to develop so-called "life-saving" medicines that are only useful in 3rd world (translated: non-paying) countries.

Socialised medicine also effectively kills incentive to develop new medicine in general, because the payout for doing such work is not worth the cost and risk.

Why do you think the vast majority of pharma's operate in the US, and ALL OF THEM consid

Well, unfortunately, we the United States without a socialized medical program have been subudizing the development of a lot of drugs for the rest world. So we're the ones paying for it $100 a pill at a time. If we don't, who will? As you've said, it takes a lot of money to create a drug. Last I checked the average figure was $800M from development to shelve. At least in the United States. Part of that also has to do with FDA requirements and the burocracy involved with running so many trials, etc..

IMO, this would be less of a problem if the United States had socialized medical care like the rest of the modern world. However, since this isn't the case the wealthier people in the US accidentally promote R&D into non live saving medicine because it suits them more, and they're willing to pay. If medical care were socialized, there'd be less of a lure to develop so many "useless" medicines, and more of a lure to develop live saving medicine.

The problem with the incentive model that you're describing is that it would need to be universally implemented or the prize has to be comparable to the profits the researching company could expect to earn from the discovery. If the incentive is tied to a clause that requires a particular pricing model, then the company will always look to see whether they can make a greater profit by declining the prize and using the same pricing models they've been using.

I'm also saddened to see that apart from HIV, there is hardly any research going on to find cures for infectious diseases

I've never understand how some people can get upset that researchers are looking into one medical problem instead of another. It seems hypocritical if they're also not doing anything to fix whatever problem they're complaining about. (Not specifically you, just people in general.)

"How dare you work on diabetes when there are children dying of malaria!" says the programmer who is working or neither diabetes nor malaria."We can give a man an erection, but we can't cure cancer?!" says the office worker who ha

Ummm some of us with diabetes did not get a "lifestyle" disease.
Some of us were born with it...

But very few are born with aids, most catch it from unprotected sex. Which is why as politically incorrect as it sounds, I dont really care if a cure is found or not. There are many other diseases that need to be cured first which arent preventable, such as cancer (yes i know smoking etc make it worse but some people get cancer having never smoked in their lives)

Think of it like treating cancer with chemotherapy. If you don't treat the person they will die. However, using this technique, if you catch it early enough, you can kill all the infected cells without killing the host. If you wait too long, the number of cells you have to kill becomes higher, and the risk of death to the host becomes higher. This is basically how chemotherapy and radiation treatment work. You kill all the cells in the affected area, and hope you don't kill the host in the process.

If the drug's targeted enough that it only kills infected cells, I don't see it killing many people from that alone. Still, HIV infects white blood cells. Killing off all the infected cells could seriously compromise an immune system even worse than AIDS.You'd probably have to stick them in an isolated ward and on all sorts of antibiotics and such to try to prevent opportunistic diseases.

Even if it only 'kills' half the cells infected, it'd probably do wonders for the average virus load of somebody infect

It may seen crude but if cutting out the HIV genes from the genome causes the infected cell to die, and killing the infected cells cause the host organism to die, then the host was essentially dead to begin with. Even with cancer, there is a point where all you can do is let it go.right now there are two options, 1. do nothing and die of AIDS in 5 or so years, 2. take expensive drugs and die of AIDS in 15-20 years; this will give people a third option, get cured or die of AIDS in 3 months.

...began with the bacterial enzyme Cre recombinase, which exchanges any two pieces of DNA flanked on either end by a certain pattern of nucleotides (DNA subunits) known as loxP.

HIV does not naturally contain loxP sites, so the team created a hybrid of the two DNA molecules, which they used to select a series of mutated Cre enzymes that were increasingly able to recognize the combined DNA.

So...this technique won't work at all in the real world. It won't even work with actual HIV even in the lab.

It's interesting research for its own sake, but in this case it has absolutely nothing to do with HIV. They simply found an interesting way to remove an arbitrary snippet of DNA. In fact, to make it work with HIV, they had to cheat and add tags to the HIV sequence.

This is like saying I could break into a bank vault after I replaced the lock with one I knew the combination to. It says nothing about the bank, only that I possess the capability to manipulate locks.

Think of this as an initial proof-of-concept. Fiddling with DNA is extremely useful - correcting genetic diseases and curing all sorts of viruses that hang out in your cells comes to mind (e.g. herpes). You could even look at curing cancer, since that's typically due to genetic mutations that could be potentially removed, making cells non-cancerous again.

Eventually, you'll want to be able to recognize and remove longer strands of DNA. I'd also worry about the efficiency - randomly removing strands of DNA from healthy cells is a good way to cause big problems. Existing gene therapies that use viruses to deliver the payload sometimes go astray and cause cancer, which is no good.

Think of this as an initial proof-of-concept. Fiddling with DNA is extremely useful - correcting genetic diseases and curing all sorts of viruses that hang out in your cells comes to mind (e.g. herpes). You could even look at curing cancer, since that's typically due to genetic mutations that could be potentially removed, making cells non-cancerous again.

No doubt. I definitely think the technique stands on its own as far as coolness factor.

What I find slightly annoying is the perceived need to validate it by linking it to HIV, which seems completely irrelevant to the actual research since the DNA segment in question could have been anything. Worse yet, it doesn't even recognize HIV at all as the headlines claim - it simply recognizes anchor groups (which HIV does not possess) and removes whatever happens to be between them. Sure, it recognizes HIV that is artificially tagged with these groups, but it would find any DNA sequence tagged with the groups. So what does this research have to do with HIV? Absolutely nothing. Seems like name-dropping to me.

I realize much of this effect is due to the funding climate in academia, which makes it impossible to get money these days unless you're coat-tailing on a handful of high-profile buzzwords. But I still find over-aggressive promotion of one's results to be distasteful. Naturally, these guys aren't the first and won't be the last.

Maybe a genetically-engineered virus designed to attack various HIV strains?

No. That's not how a virus works. A virus, outside of a living cell is a inert bunch of proteins, nucleic acids and sometimes lipids. A "genetically-engineered" virus could only work if it would infect the same cell as the HIV. If two different viruses infect the same cell, a process called interference can occur. This can screw both of the virus types, but the cell is screwed as well. And to kill all of the HIV infected cells, we would need the engineered virus to be more infective as HIV. So essentialy

It may be 'cheating' but it does prove that the process can be done.
It proves that one can cut HIV, albeit a prepared version, out of DNA
If the procedure can be done under perfect lab conditions, it can be tweaked and changed to work outside the lab
A major accomplishment if you ask me

The final enzyme did work with real HIV in the lab. They identified a site in HIV similar to the cre binding motif, but which cre was not able to bind. They created intermediate sequences to bridge the gap between the cre binding site and this HIV sequence. Using directed evolution they could evolve cre to bind sites progressively more unlike the cre site and progressively more like the HIV site. The final outcome was an enzyme able to excise sequences flanked by the HIV specific pattern.

The final enzyme did work with real HIV in the lab. They identified a site in HIV similar to the cre binding motif, but which cre was not able to bind. They created intermediate sequences to bridge the gap between the cre binding site and this HIV sequence. Using directed evolution they could evolve cre to bind sites progressively more unlike the cre site and progressively more like the HIV site. The final outcome was an enzyme able to excise sequences flanked by the HIV specific pattern.

One would assume that there are a few critical sequences in the virus, without which it would not function or evolve around. Could the structure of its protein shell be corrupted to cause it to immediately fall apart, a la penicillin? Could changes be made to ensure that it would remain forever dormant?

It would seem that, with this technique, a little sabotage might get nearly the same benefit as cleaning it all out, for much less effort and risk.

They identified a site in HIV similar to the cre binding motif, but which cre was not able to bind. They created intermediate sequences to bridge the gap between the cre binding site and this HIV sequence.

And now that the concept is proved they can do the same, perhaps with more steps, for a strongly-conserved section of HIV's genome and make an enzyme that targets all, or a very broad range of, HIV strains.

Another question is how much of the HIV DNA would you have to remove to render it inert? Or hell, just make it produce bad (as in neutered) copies in cells? Surely nearly all strains of HIV have some fundamental part of the genome in common. If so, this would be potentially very helpful (in the distant future)

Surely nearly all strains of HIV have some fundamental part of the genome in common.

Nothing which is unique to them, AFAIK. This is a large part of the problem indeveloping said vaccine: HIV (like most viruses) has a small genome (9 genes),but it also mutates rapidly. In the course of an infection you will develop newstrains yourself i.e; drug resistance.

Although I feel this may also be "cheating", as Mr. Underbridge points out, I don't care. It gets us in the door, allows us to wedge it open, and take out what we want. I look it it more like "painting" a tank with a laser target so the smart-bomb knows where to strike. This is still a pretty good milestone. Maybe, just maybe, in my lifetime, we'll see this disease destroyed. I would like to live to see that.

No, it's not like painting a tank with a laser... it's like hanging a bomb over the tank using a crane.All they demonstrated was that if you add LoxP sites to a DNA sequence, you can then cut the segment out using Cre recombinase - something the scientific community was doing for a decade now, when we design conditional knockouts. But just cause the DNA sequences happened to be HIV, this is now ground-breaking news?

A good analogy would be an article about a new way to identify Iraqi insurgents among the civ

This is a big deal because it shows that this technique which has been used for years to cut out fragments of the genome for replication (via PCR and other methods) could be used to remove the viral elements from a genome. It's a big deal research-wise, but the major problem that will hinder this application from practical application is that HIV hybridizes EXTREMELY fast. Using an artificial bacterial enzyme to remove dna fragments requires a specific nucleotide sequence that it targets. Since HIV "cha

DNA evidence isn't as "rock solid" as you imagine. What they do is chop up the DNA just like the article did and measure the lengths, so at best it's a matter of statistics, at worst it's garbage-in, garbage-out.

It took longer than usual for one of you technological pessimists to pipe up:)My guess is that you couldn't change too much of your own DNA without messing yourself up pretty badly. If people started using this as a masking technology, you would have to change the testing method to only include genes that you can't really touch, or maybe just screen for telltale enzymes of genetic manipulation. In the end it would be like trying to bleach off your fingerprints.

Hey, I like technology as much as anyone, but history has shown that we have to think of everything before making it possible to do some things with technology before others are possible.

DNA is one of thoose things that if tamperable to early, could change legal, medical, & who knows what else-al systems in ways we haven't even begun to imagine before it even became beneficial to do it.

On a brighter note, perhaps if robots completely ran humanities production & DNA could easily be altered to i

This weeks Escape Pod Podcast [escapepod.org] (hosted by Steve Eley) is called the Giving Plague and touches on viruses, HIV, and the potential symbiotic relationship and borg like integration viruses can have with Human cells.

One of the thoughts is that viruses actually benefit the race in the long term, as we will eventually form a symbiotic relationship with the majority of them. (uses e-coli in our gut as an example), but how one day someone will be resistant to AIDS and that will make the human race stronger.

To go even further, e.coli that naturally exists in your gut is only beneficial because it is uninfected with a bacteriophage (a virus that infects bacteria) that turns e.coli into a dangerous form that causes food poisoning (such as the cases of e. coli you hear about from undercooked meat).

well considering that when you take a really good strong antibiotic that kills the E. Coli in you colon you feel like Mike Tyson has been using your belly for a heavy bag and that you would die with out the vitamin K that the E. Coli produces, I'd say it was pretty well assimilated.

It is believed that a variation of CD4 confers resistance to the bubonic plague as well as HIV.There are supposedly communities in Europe (the UK IIRC) where this was selected for during theblack death.

Ok, this is my wild idea.We know that we can "reboot" the immune system by destroying the bone marrow and repopulating it with new one coming from a donor.

Now lets say that we do an autotransplant. First we take a sample from the donor and then this sample is treated with the enzyme so all of the HIV's DNA is removed. Next, we introduce a gene on this cultured cells that will produce the enzyme, thus rendering them immune to infection. Next we destroy the donor's bone marrow and implant the new one.

This idea is based on a widely disseminated misconception, that T-cells don't reproduce when out of bone marrow. They do, and happily so, after being activated by other cells, antigens, cytokines and a bunch of other means. Your method has been tried, in a way. A patient's blood was essentially flushed with healthy blood from donors, so his whole blood was exchanged. It did no good in the long term, because the HIV infects also macrophages in other tissues than blood. The next wave of the infection came from those macrophages.

But this is based on the idea that the new T cells coming from the bone marrow would be immune to infection. Eventually they would be the only ones around and the HIV wouldn't have room on them. Can the HIV live in a host where it can't infect the T cells?

I am going to play devil's advocate here..I could be wrong (super likely)..and please understand I'm not dissing.. it just means I am misunderstanding you, or you need more research on it, or suggest different ideas.Couple of negatives that will have to be worked around/disproven:

1) Physiological response to that enzyme may not be good + will need a reliable turn off/turn on dosage mechanism (prolly not a big deal?)2) Remaining viruses may have mutant offspring with a different HIV-01 LTR sequence than wh

They've done it in vitro in a lab. Which is a good start, but that doesn't mean you can now safely screw anything that walks.

They probably haven't developed anything which they could conceivably be administered to a living organism yet - let alone tried administering it to one. Then you've got a battery of tests to make sure it's safe and effective - there's probably at least another 10 years before this could really be a treatment.

The great majority of potential treatments never make it through that development/testing process.

This approach is not really that useful in terms of coming up with a cure for HIV. The reason is that the complexity to excise a specific sequence of that length is WELL beyond our knowledge.

But this will hopefully lead to a real "cure" in being able to mutate it to being none-lethal or even quiescence. It struck me back in the early 80's, that about the only way is to insert a virus inside this virus and break the formation of the protein chain.

... it would be move valuable than gold! If you could suddenly change peoples dna, not only would this be dangerous as to where it goes, but it could be invaluable. People who have genetic diseases could all be cured, by replacing parts of their dna. People who wanted sex changes could have their dna changed also. The scary part is that people who did not like a certain race of people could change that race genetically. The possiblities are endless.

No. not like this. Sex is determined by the presence of a whole chromosome,not a piddly gene. Furthermore, swapping out chromosomes in somatic cells isn'tgoing to cause your penis to fall off.

The scary part is that people who did not like a certain race of people could change that race genetically. The possiblities are endless.

Again, not quite. There are some markers that strongly suggest various aspects oflineage, but nothing definitively proclaiming "I am Czech!" (Yes, that's ethnicity, not race. But the idea is the same for this nefarious purpose) For that matter, manypeople whom you might not suspect of having said markers will possess

Cre is an bacterial enzyme (a member of a family of enzymes called site specific recombinases) commonly used by researchers attempting genetic manipulations of dna. The cre enzyme recgonises a specific dna sequence (called LoxP sites) just over 30 letters (base pair) long and then catalyses a reaction which can either cut out dna, insert dna or reverse the orientation of dna flanked by loxp sites (precisely what the cre enzyme will do depends upon the number of sites and the order and orientation of the sites).
The HIV virus does not contain LoxP sites so these guys "evolved" the cre enzyme by a selective process to recognise DNA sequences that were initially a hybrid of a part of the HIV virus sequence and the cre Loxp site. they continues this evolution until a modified Cre enzyme (now called Tre) could actually recognise the original HIV dna sequence.
They then used this Tre enzyme to cut out the HIV virus dna that had inserted itself into the cell genomic dna, freeing the cells of the HIV virus.
This is a pretty interesting article, however, as the authors state this is preliminary work. One problem i can envision stems from the fact that HIV virus often inserts itself numerous times into the host genome. When researchers are using cre they have to be careful about the number of copies of the Loxp site in the genome or it is possible for the cre enzyme to cause large deletions of genomic dna or even cause translocations (when the genomic dna found on one chromosome is erroneously attached to that of another chromosome). Such changes to the dna can be highly deleterious to the cell and initiate cancerous changes.
hope this helps.

What exactly is this "Tre" then? I see it's an enzyme, but I guess I'm still a bit confused. Cre acted as a catalyst to procure a specific reaction for a specific DNA sequence? Is that an attribute of the chemical composition of the enzyme, or, well...I guess I really don't understand where that came from. Is it a specific enzyme, or is "Cre" the name attributed to ANY enzyme that acts in this way?

Awesome explanation. The story didn't make any sense to me until I read your explanation of evoloving Cre into something useful for this purpose. Thank you. This is the most nerdly thing I've read all day.But I have to say that I disagree about needing to be careful about the number of infections in the host cell. HIV infects differentiated cells that do not naturally reproduce, so mutagenesis leading to cancer is unlikely, and killing infected cells is very nearly as useful as curing them. The body ca

Well then all we need to do is write a simple script to iterate over all of the cells in the body and run the sed command. The list may be too long though, so we might have to use xargs, but that's no big deal.

"These German dudes ripped HIV out of your cells after it already got into your DNA. OMG! They think they might can cure AIDS with this. They're not too sure if it'll work, because it's gonna be WAY harder if you have AIDS real bad. 'The German dudes... started out with this enzyme that'll swap out any DNA it finds that has these certain two nucleotide thingies on the ends of 'em with these mutant ones that they whipped up in the lab. Then this other one named Tre comes in, kicks ass, and takes names. After about 3 months, bye-bye, HIV! They were talking about it today in Science'"

Sox2 explained it pretty well, I'll try to dumb it down even more. Certain enzymes recognize patterns on DNA, and chop them right at that location. HIV is a virus that inserts itself into your DNA (unlike most viruses, which just use your cells resources to reproduce). The scientists evolved an enzyme that recognizes the sites on either side of the HIV, chops them up, and splices them back together--effectively removing the HIV.
The reason it's "preliminary work" and not a magical cure is because the 'patterns' recognized by the enzymes (and the enzymes themselves) were morphed into "fake" sites. In actuality, it's much harder to create enzymes that recognize the actual patterns of the HIV. In addition, these enzymes might find patterns elsewhere in DNA and accidentally chop up your DNA in the wrong locations, removing important parts of your DNA.

I really wouldn't worry about that. Sure, it could potentially make a weapon, if properly worked upon with that goal in mind. You would have to be pretty dedicated though. And it would have the side effect of attacking all humans because of the similarities of DNA and the probabilities of mutation... who would want to create a weapon to kill everyone on the planet when we already have so many. Smallpox would make a far more viable weapon, or an old fashioned cobalt bomb.

Let's see - if I understand correctly, we've developed the capability to engineer something that can go in and ERASE very specific segments of people's DNA?****

Trust me, you're not wrong here. Someone will develop this into a bio-weapon. Want all corn crops blighted? Kill off all the bees? Or just make all males that are affected sterile. Or induce madness or... The potential nightmares of nanotech in the wrong hands is truly appalling.

While I normally would not dignify such a comment with a reply, I must state this: if you are ignorant of how high heterosexual HIV infection rates are around the world, then you are simply to be pitied.